1. Field of the Invention
The present invention relates to a method, composition and element for the determination of neuraminic acids in aqueous liquids.
2. Description of the Related Art
Biological fluids from the human body contain various amounts of sialic acids, which are a class of neuraminic acids. These acids are important in cell-to-cell interaction, as hormone receptor sites and as immunodeterminants for cells. Various pathological states, including cancer, have been found to accompany increased serum content of sialic acid.
Recently, a relationship between the level of sialic acid in saliva and the period just prior to ovulation in human females has been found. This indicates a potentially important diagnostic role for the assay of sialic acid.
Various colorimetric chemical methods have been described to measure sialic acid, including resorcinol, orcinol and thiobarbituric acid procedures. These methods are not absolutely specific for sialic acid, however, as other carbohydrates interfere with the color development in these procedures.
A more specific method for assaying for sialic acid is described in Burnetti, Swanson and Roseman, "Methods in Enzymology", 6, pages 465 to 473 (1963). This method comprises submitting a sample to acid hydrolysis to release the sialic acid which is bound to a more complex biological material in the fluid. The freed sialic acid is then reacted with N-acetyl neuraminic acid aldolase to form N-acetyl mannosamine and pyruvate, which is reacted with lactic dehydrogenase (LDH) and reduced nicotinamide adenine dinucleotide (NADH) to measure the resulting decrease in NADH spectrophotometrically at 340 nm. However, N-acetyl neuraminic acid aldolase preparations in the assay contain NADH oxidase which interferes with the assay in that it oxidizes NADH to NAD.sup..sym., thereby introducing a source of positive bias.
Additionally, it is desirable to use dry elements to assay for sialic acid, since a structure containing reactants in a coating would greatly simplify the assay. NADH is quite unstable during coating and storage, is expensive and its limited absorbtivity produces low sensitivity at low analyte levels. Thus, a dry element containing NADH as a reactant would not be advantageously used.
An alternative method for measuring the pyruvate generated by the aldolase (and thus the sialic acid) is highly desirable.